Optimized preservation of extracellular matrix in cardiac tissues: Implications for long-term graft durability

Background. Cryopreservation of human tissues, particularly heart valves, is widespread in clinical practice although the effects of this process on underlying tissue structures and its potential impact on valve durability have been poorly studied. Multiphoton imaging and second-harmonic generation (SHG) microscopy permit high-resolution, noninvasive analysis of living tissues at a subcellular level. In the present study we used these novel imaging modalities to compare the effects of vitreous and frozen cryopreservation on the extracellular matrix (ECM) of cardiac tissues. Methods. Conventional histology, electron microscopy, and multiphoton imaging to obtain autofluorescence and SHG images were performed on cardiac tissues to characterize the ECM in fresh, vitrified, and frozen cryopreserved tissues. Results. Autofluorescence and particularly SHG images revealed that conventional frozen cryopreservation of cardiac valves, when compared with fresh or vitrified tissues, leads to the loss of normal ECM structures in valve leaflets. Similar results were found in all other cardiac tissues suggesting that structural deterioration of the ECM is a common consequence of frozen cryopreservation. Conclusions. Our results demonstrate that conventional cryopreservation, when compared with fresh or vitrified tissues, causes more destruction of normal ECM structure, which might contribute to eventual graft dysfunction. Whether vitrification preservation will translate into greater durability or less valve failure will need to be determined.